Project Summary/Abstract The goal of the proposed Phase I research is to develop a set of commercial kits for footprint-free genome engineering. The core technologies that serve as the foundation of these kits are based on the CRISPR/cas system, which was originally identified in bacteria and recently shown to also function in mammalian cells. The main advantage of the proposed gene targeting system is that the specificity of the target DNA sequence is dictated by a guide RNA, which can base-pair with one strand of double-stranded DNA by forming an R-loop. Currently, zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) provide possibilities to specifically and permanently modify the human genome. There are commercial reagents and services available for these two technologies. However, these two technologies are based on protein-DNA recognition, which requires designing and creating new enzymes every time a unique DNA sequence is to be targeted. The assembly of new ZFNs or TALENs is cumbersome and costly, and is presently only performed by a small number of laboratories or through expensive commercial services. Successful execution of the proposed project will establish a fully tested, market-ready system for making any desired changes to mammalian chromosomes. The proposed kits can be easily adopted by any lab at significantly lower costs compare to the ZFN and TALEN services or self-assembled processes. Opposite to the recently published vectors that delivery either the cas nuclease or the guide RNA, the proposed system is in an all-RNA format. The mRNA system for expression of the required enzyme works by expressing through an inherently short-lived vector which is impervious to genetic recombination and the problematic aspects of vector replication are sidestepped because mRNA transcripts are simply resupplied via culture media for the time required to achieve desired level of chromosome modifications. These properties, in conjunction with the excellent expression efficiency and kinetics which have been demonstrated in mRNA reprogramming of human fibroblasts, make the system a good candidate for a successful commercial kit.